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14 protocols using hfdpc

1

Culturing Follicle Dermal Papilla Cells

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HFDPC were purchased from PromoCell (Heidelberg, Germany). The cells were maintained in follicle dermal papilla cell basal medium supplemented with 4% fetal calf serum, 0.4% bovine pituitary extract, 1 ng/mL basic fibroblast growth factor, and 5 μg/mL recombinant human insulin (PromoCell) at 37°C in 5% CO2.
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2

Cell Culture Conditions for Various Cell Lines

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Mouse skin melanoma cells (B16F10; JCRB no. JCRB0202) and human fibroblast cells (JCRB no. JCRB1006.4F) were purchased from the JCRB Cell Bank (Osaka, Japan). Murine RAW 264.7 macrophage cells and human prostate cancer cells (DU-145) were purchased from the American Type Culture Collection (Rockville, MD, USA). Primary human follicle dermal papilla cells (HFDPC) were obtained from Promo Cell GmbH (Heidelberg, Germany). The B16F10 cells were grown in a MEM culture medium supplemented with 10% FBS and 1% penicillin/streptomycin. The RAW 264.7 cells were grown in a DMEM culture medium supplemented with 4500 mg/L D-glucose, 10% FBS, and 1% penicillin/streptomycin. The DU-145 cells were grown in RPMI-1640 culture medium supplemented with 10% FBS and 1% penicillin/streptomycin. The fibroblast cells were cultured in a DMEM culture medium supplemented with 10% FBS, and 1% penicillin/streptomycin. The HFDPC cells were cultured in a Growth Medium Kit supplemented with 10% FBS and 1% antibiotic-antimycotic 100× solution. All cell lines were maintained under a humidified atmosphere containing 5% CO2 at a temperature of 37 °C.
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3

Wnt10B Modulation of Dermal Papilla Cells

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HFDPCs isolated from human dermis originating from the lateral scalp were purchased from PromoCell GmbH. Cells were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.; 10099-14-FBS) and 1% Penicillin-Streptomycin Solution (E607011, Sangon Biotech Co., Ltd.), at 37°C and 5% CO2. Human recombinant WNT10B protein was purchased from R&D Systems (cat. no. 7196-WN). The WNT10B treatment conditions used in the present study were as previously described (38 (link)). WNT10B protein was reconstituted in PBS containing 0.1% bovine serum albumin (BSA; Sangon Biotech Co., Ltd.; A602440) to form a solution of 10 µg/ml and used at a final concentration of 1 µg/ml. The expression of the Wnt target gene β-catenin in DPCs was increased following treatment with 1 µg/ml WNT10B protein for 3 days at 37°C, which was in agreement with the results of a previous study (25 (link)) indicating that the culture conditions were appropriate.
For RNA-sequencing (RNA-seq), the DPCs were divided into two groups: i) The experimental group, in which DPCs were cultured in DMEM supplemented with 10% FBS and 1 µg/ml recombinant WNT10B for 3 days at 37°C; and ii) the control group, in which DPCs were cultured in DMEM containing 10% FBS and 1 µg/ml BSA for 3 days at 37°C.
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4

Cytotoxicity and Proliferation of Hair Follicle Cells

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In vitro cytotoxicity and cell proliferation studies on normal hair follicle dermal papilla cells were undertaken as described previously [33 (link)]. Cultured human hair follicle dermal papilla cells (HFDPCs) (Promo-Cell, Heidelberg, Germany) from passage 4 were seeded (7.5 × 104 or 3.75 × 104 per mL) onto a 96-well plate (Nunc, Wiesbaden, Germany) and grown to a confluence of 60–70% for 24 h. The MTT assay for cytotoxicity and proliferation was conducted for 5 days. Treatment was conducted with dutasteride alone (dutasteride was dissolved in DMSO at 100 mM and serial dilutions were prepared from 100 to 1.6 µM), dutasteride in NLCs uncoated or coated with 5% CSO-LA (dutasteride concentration 12.5–100 µM), and empty NLCs uncoated and coated with CSO-LA, with minoxidil (100 µM) as a positive control. Absorbance was determined spectrophotometrically at 570 nm using a microplate reader (SpectraMax®M2e, Mol. Devices, San Jose, CA, USA). The results are expressed as percentages of untreated controls in four cultures. The reported values are the means ± SD.
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5

Anthocyanin-rich BCE Powder Effects on HFDPCs

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BCE powder, CaNZac-35, was purchased from Koyo Mercantile Co. (Tokyo, Japan). BCE contained high concentrations of anthocyanins (38.0 g/100 g BCE) [11 (link)]. HFDPCs derived from the temples of Caucasian middle-aged women were obtained from PromoCell (Heidelberg, Germany). Cells were maintained in follicle dermal papilla cell growth medium (PromoCell). All cell culture experiments were conducted at 37 °C in a humidified incubator containing 5% CO2.
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6

Culturing Human Hair Follicle Cells

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Human hair follicle dermal papilla cells (hFDPCs) were purchased from PromoCell
(Heidelberg, Germany). The hFDPCs were cultured in FDPC growth medium
supplemented with 4% (v/v) fetal calf serum, 0.4% (v/v) bovine pituitary
extract, human recombinant basic fibroblast growth factor (1 ng/mL), and human
recombinant insulin (5 μg/mL; PromoCell). Human hair germinal matrix
cells (hGMCs) were obtained from ScienCell Research Laboratories (Carlsbad, CA,
USA). The hGMCs were cultured in mesenchymal stem cell medium supplemented with
5% (v/v) fetal bovine serum, and 1% (v/v) mesenchymal stem cell growth
supplement (ScienCell Research Laboratories). Both hFDPCs and hGMCs were
cultured at 37°C in a humidified atmosphere of 5 % CO2.
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7

Modulation of Human Hair Follicle Cells

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Human follicle dermal papilla cells (HFDPCs) (PromoCell GmbH, Heidelberg, Germany) were cultured in keratinocyte serum-free medium (PromoCell GmbH) with supplied supplement and 100 mg/mL penicillin and streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO2 incubator at 37°C. Cells were used within ten passages for the experiment. The HFDPCs (1.5 × 105 cells/mL) were seeded in collagen type-I coated 96-well and 6-well plates and incubated overnight. The cells were treated with DHT (10 nM, Sigma-Aldrich, St. Louis, MO, USA) with or without finasteride (1 μM, Sigma, USA) or 50 μg/mL of S. hexaphylla extract for 24 and 72 h. Cellular morphology was observed at 200 magnification using a phase contrast microscope (Olympus, Japan).
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8

Isolation and Culture of Human Hair Follicle Dermal Papilla Cells

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Human hair follicle dermal papilla cells (HFDPCs) (Promo-Cell, Germany) were grown in DPC growth medium (Promo-Cell, Germany) containing 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL, MD) in 5% CO2 at 37°C.
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9

Cultivating Human Follicle Dermal Papilla Cells

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Human follicle dermal papilla cells (HFDPCs) were purchased by PromoCell. The cell line was cultured in a specific medium (follicle dermal papilla cell growth medium-ready to use with its supplement mix, PromoCell) in conditions of complete sterility and maintained at 37 °C with 5% carbon dioxide (CO2) atmosphere. The product HYDRO DELUXE BIO was weighed and dissolved at the concentration of 80 mg/ml in a complete cell culture medium.
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10

Cicaria Fraction Modulates Hair Growth

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To evaluate the effect of the Cicaria fraction on the expression of hair growth factors, a genetic analysis of the hair growth factors was conducted in HFDPCs. First, HFDPCs were purchased from PromoCell (Heidelberg, Germany) and cultured in a DPC growth medium containing the supplement and 100 U/mL of penicillin and 100 ug/mL of streptomycin. For gene expression analysis, HFDPCs were inoculated in a 6-well plate at a density of 3 × 105, and then cultured for 24 h in an incubator at 37 °C and 5% CO2. After 24 h, the culture medium was discarded, washed with DPBS, and replaced with a medium not containing supplements or a medium containing 10 ppm Minoxidil, as a positive control, or Cicaria fraction 1 ppm, and further cultured for 24 h. Subsequently, RNA was isolated from cells using an RNeasy Mini Kit (Qiagen); RNA was quantified at 260 nm using nanodrop, and the cDNA was synthesized in amplifiers (C1000 Thermal Cyclers, Bio-Rad, USA) using 2 μg of RNA. The degree of VEGF, FGF7, and HGF gene expression was finally evaluated by performing a real-time polymerase chain reaction in a real-time PCR machine using a mixture of FGF7 and HGF primers and cyber green (SYBR Green Supermix) to synthesize cDNA. Primer sequences and reaction conditions are shown in Table 1, and the expression levels of genes were analyzed relative to the β-actin gene.
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